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Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. An Author Correction to this article was published on 16 October The amygdala processes positive and negative valence and contributes to addiction, but the cell-type-specific gene regulatory programs involved are unknown. We generated an atlas of single-nucleus gene expression and chromatin accessibility in the amygdala of outbred rats with high and low cocaine addiction-like behaviors following prolonged abstinence. Differentially expressed genes between the high and low groups were enriched for energy metabolism across cell types. Differences in chromatin accessibility between high and low AI rats implicated pioneer transcription factors in the basic helix-loop-helix, FOX, SOX and activator protein 1 families. We observed opposite regulation of chromatin accessibility across many cell types. Most notably, excitatory neurons had greater accessibility in high AI rats and inhibitory neurons had greater accessibility in low AI rats. The amygdala regulates numerous behaviors related to emotions, motivation and memory 1 and is implicated in various neuropsychiatric disorders including addiction 2 , 3. Repeated drug use engages the amygdala to form drug-associated memories and reinforces drug-seeking behavior 4. In addition, during withdrawal from addictive drugs, the amygdala mediates negative emotional states, such as anxiety, fear and irritability 4. Avoidance of these aversive emotions enhances the incentive value of the drug, leading to sustained drug-seeking behaviors and relapse 5 , 6 , 7. The amygdala is composed of several interconnected subregions 8 including the basolateral amygdala BLA and the central amygdala CeA 9 , While the behavioral function and connectivity of the amygdala have been established 1 , the role of distinct neuronal and non-neuronal cell subpopulations in addiction remains unclear. Recently developed single-nucleus RNA-sequencing snRNA-seq and single-nucleus assays for transposase-accessible chromatin snATAC-seq have enabled the study of the cellular function and diversity of the human, mouse and nonhuman primate brains 11 , 12 , 13 , 14 , 15 , 16 , However, their application to study the neurobiology of addiction has been limited. However, these previous studies used inbred rodent strains, which limited examination of genetically mediated differences in susceptibility to addiction-like behaviors. Furthermore, these studies focused on acute drug treatments and therefore did not explore molecular changes that accompany long-lasting addictive-like behaviors. To address these limitations, we performed snRNA-seq and snATAC-seq using amygdala tissue from outbred heterogenous stock HS rats obtained from a large genetic study of cocaine addiction-related traits These rats were exposed to extended access drug intravenous self-administration IVSA 24 , 25 , IVSA is linked to neurochemical changes in key brain regions, such as those observed in humans with cocaine use disorder HS rats were used because they have high levels of genetic variation and rich phenotypic diversity 28 , 29 , 30 , The animals were trained to self-administer cocaine in operant chambers via lever press fixed ratio of 1 with 0. We measured the number of cocaine rewards, or lever presses, during each session of the behavioral protocol. Escalation of intake was determined as the increase in the mean number of cocaine rewards during LgA sessions compared with the first day of the LgA phase. Motivation for cocaine was assessed at the end of ShA and LgA phases, using a progressive ratio PR schedule of reinforcement, where the number of lever presses required to obtain a cocaine infusion increased progressively. For each rat Fig. Error bars in d — g represent s. We classified rats into high and low AI groups Fig. These results show that our model of extended access to cocaine IVSA in outbred rats captures several relevant aspects of cocaine use disorder. Visualization of the integrated data indicated that the clustering is not influenced by batch effects such as sequencing library, percentage of mitochondrial DNA or individual rats 36 Supplementary Fig. Using established cell-type-specific marker genes 11 , 37 , 38 , 39 , 40 , we annotated the snRNA-seq clusters Fig. We also identified seven subtypes of inhibitory neurons based on the expression of known cell marker genes Fig. Cell-type proportions seemed to be consistent across samples Supplementary Figs. The total number of nuclei we obtained for each cell type varied substantially Fig. For most downstream analyses, we focused on the six most common main cell types Fig. Data are combined across 19 samples, with high, low and naive AI labels. Cells are colored by cluster assignments performed with KNN analysis. We assigned cell-type labels to clusters based on the expression of known marker genes. The shading and diameter of each circle indicate the estimated mean expression and the percentage of cells in the cluster in which the marker gene was detected. Cell clusters from the CeA and BLA were distinct from one another, but these regions collectively contained most cell types identified in the whole amygdala Supplementary Fig. Consistent with the known cell-type composition of the CeA and BLA 42 , cell clusters from the CeA coclustered primarily with inhibitory neurons whereas those from the BLA coclustered with excitatory neurons Supplementary Fig. Glial cell types from the whole amygdala contained cells from both subregions, except for astrocytes, which coclustered mostly with cells from the CeA but not those from the BLA, suggesting that astrocytes might play a specific role in CeA-related function Supplementary Fig. The snRNA-seq and snATAC-seq datasets we generated are the first single-cell atlas of molecularly defined cell types in the rat amygdala under normal conditions and during cocaine addiction-like behaviors. We used the negative binomial test to identify differentially expressed genes DEGs between high and low AI rats in each cell type Fig. Fractions indicate the number of bootstrap iterations in which the log 2 FC estimate was significantly different from 0. Boxplot hinges are the 25th and 75th percentiles; whiskers extend to the minimum and maximum; center line is the median and dotted line is the mean. In total, we identified unique significant DEGs with large effects in at least one cell type and 8, unique significant DEGs with small effects in at least one cell type. These DEG could reflect inherited differences in gene expression that predate exposure to cocaine, or they could be caused by differences in the amount of self-administered cocaine. For example, Kcnq3 was differentially regulated across neuronal and glial cell types, and encodes a subunit of a potassium channel implicated in the regulation of reward behavior and susceptibility to drug addiction Fig. Additionally, Fkbp5 and Sgk1 , two transcriptional targets of the glucocorticoid receptor, were differentially regulated in glial cell types and are associated with reward behavior and drug addiction vulnerability Fig. To further examine the contribution of genetics to observed differences in gene expression, we leveraged genotypes and gene expression data from a reference population of drug-naive HS rats This allowed us to predict gene expression based on cis-genetic variation in the absence of cocaine exposure. Specifically, we trained models to predict gene expression from single nucleotide polymorphism genotypes 51 using whole-brain bulk RNA-seq from naive HS rats, and estimated the fraction of variance in expression that was explained by cis-genetic variation r 2. We used the trained models to predict the expression of genes with at least one cis-acting eQTL 8, genes for each of the rats in our snRNA-seq dataset and compared the differences in mean predicted expression in high versus low AI rats with the observed differences in expression for each cell type after filtering out genes with low r 2 Supplementary Table 3. These observations indicate that genetic differences in high versus low AI rats contribute to some of the observed differences in expression. Cocaine exposure probably also plays a role; however, quantifying the relative contributions of cocaine and genetics is challenging due to limitations in the genetic predictions of gene expression. We identified significant enrichment of several pathways related to addiction, including neurotransmission and energy metabolism Fig. Most cell types showed enrichment of genes belonging to the oxidative phosphorylation pathway, which, together with glucose metabolism, is the main energy source for synaptic activity and action potentials 53 , These observations suggest that addiction-like behaviors are associated with alterations in the metabolic state of amygdalar cell populations, which can directly impact neural network activity in the amygdala. To test the hypothesis that altered cellular metabolic state impacts neural activity in the amygdala, we focused on GABAergic transmission, which has been implicated previously in addiction 2. These results support the hypothesis that the cocaine addiction-like behaviors in high AI rats reflect increased GABAergic transmission. Electrophysiological recordings were taken before and after pBBG treatment from tissue slices of five naive, five low AI and five high AI rats. Rats with low and high AI were injected with vehicle or pBBG following a period of prolonged abstinence, and re-exposed to SA chambers in the absence of cocaine. Error bars in panels b , c , and h represent s. To further investigate the link between GABAergic transmission and energy metabolism in the amygdala with cocaine addiction-like behaviors, we measured the frequency and amplitude of sIPSCs before and after application of S-bromobenzylglutathione cyclopentyl diester pBBG 55 , The above results led us to hypothesize that GLO1 inhibition might reverse behavioral differences observed following prolonged abstinence from cocaine IVSA. Rats were subjected to the same operant conditions of cocaine IVSA but without drug availability, and reinstatement was triggered by re-exposure to the cocaine infusion-associated light cue. We used MACS2 ref. The pseudobulk chromatin accessibility showed the expected cell-type-specific patterns at the transcription start sites TSS of marker genes for each cell type Fig. This indicates that the snRNA-seq and snATAC-seq results are consistent and that gene expression changes are associated with changes in promoter chromatin accessibility. There are many cases where motifs display increased activity in upregulated peaks in neurons while also displaying decreased activity in downregulated peaks in oligodendrocytes. The differential peaks were categorized into those with higher upregulated or lower downregulated accessibility in the high AI rats Supplementary Fig. Astrocytes had roughly equal numbers of up- and downregulated peaks, but other cell types showed profound directional biases. These biases probably reflect altered activity of transcription factors TFs controlling large transcriptional programs. These findings confirm that the differences in chromatin accessibility and gene expression are concordant. In total, 3. This enrichment may indicate that changes in chromatin associated with addiction-like behaviors are more concentrated at promoters, or that we have greater statistical power to detect changes at promoters, due to larger effect sizes or greater chromatin accessibility. We hypothesized that differences in chromatin accessibility between high and low AI rats are caused by differential TF activity. As many TFs recognize similar motifs, we grouped them into motif clusters and summarized results across cell types Fig. The motif cluster with the most significant difference in accessibility between high and low AI rats contained motifs for basic helix-loop-helix bHLH TFs. This motif cluster had substantially higher accessibility in the excitatory neurons of high AI rats compared with low AI rats deviance 3. Thus, the widespread increases in chromatin accessibility in excitatory neurons of high AI rats could reflect increased activity of pioneer TFs that recruit chromatin remodelers. However, we did not observe corresponding upregulation in the expression of genes encoding TFs belonging to these clusters Supplementary Data 5 and Supplementary Data 12 , suggesting that a different mechanism might affect their activity. Many motif clusters with increased accessibility in the neurons of high AI rats have decreased accessibility in oligodendrocytes Fig. AP1 and MEF2 motifs are implicated in addiction 64 , 65 , 66 , 67 and their expression changes in the brain following chronic exposure to cocaine and other drugs 68 , 69 , 70 , 71 , These results implicate many motif clusters associated with addiction-like behaviors across thousands of regulatory regions and in a cell-type-specific manner. To assess whether our rat snATAC-seq data is relevant for human addiction-related traits, we mapped the accessible chromatin peaks to the human reference genome and performed cell-type-specific linkage disequilibrium LD score regression 73 using summary statistics from well-powered genome-wide association studies GWAS for alcohol and tobacco use 74 , These results indicate that the regulatory architecture of HS rats is relevant for human addiction-related traits. To better understand the molecular basis of addiction, we generated an atlas of single-cell gene expression and chromatin accessibility in the amygdala of rats with divergent cocaine addiction-like behaviors after a prolonged period of abstinence. Our dataset is the largest resource of cell types in the mammalian amygdala, with over , nuclei in our snRNA-seq dataset and 81, nuclei in our snATAC-seq dataset Fig. The snATAC-seq dataset is the first map of cell-type-specific regulatory elements in the amygdala, enabling the identification of TF motifs that may drive addiction-related processes. Previous rodent snRNA-seq studies have focused on the acute effects of passive treatment with psychoactive drugs 22 , 23 , which cannot fully capture the motivational processes underlying addiction. In contrast, our behavioral protocol using extended access to cocaine IVSA reflects key aspects of cocaine addiction, including escalation of drug use, enhanced motivation for drug seeking and taking, and persistent drug use despite adverse consequences In addition, using an outbred rat population allowed us to correlate molecular differences not only with a high AI phenotype, which reflects vulnerability, but also with a low AI phenotype, which reflects resiliency to developing addiction-like behaviors One striking finding from our study is that there were strong biases in the direction of regulation of open chromatin regions between high and low AI rats in several main cell types Supplementary Figs. Most of these differences were small, suggesting that the combined action of many small effects on gene expression and chromatin accessibility underlies the behavioral differences between rats with high and low AI. Because the HS rats are genetically diverse, the molecular differences between high and low AI rats could arise from genetic differences or it could be a consequence of consuming different amounts of cocaine. The results are consistent with a polygenic model wherein addiction-like behaviors result from the collective action of a large number of genetic risk loci with small individual effects. This is a plausible explanation because of the high genetic diversity in the HS rats and because complex traits, including addiction, are known to be highly polygenic in humans 73 , Alternatively, a relatively small number of TFs could affect many downstream genes and chromatin sites. Because the motifs with the strongest chromatin accessibility differences Fig. These explanations are not mutually exclusive, and it is probable that some differences are caused by eQTLs while others are caused by differences in the activity of upstream regulators which themselves may be affected by genetics or other factors. To uncouple pre-existing genetically controlled gene expression differences from cocaine-induced neuroadaptations, we compared our observed DEGs with differences in expression obtained from genotype-based prediction models. We found significant correlations in observed versus predicted differential gene expression between high versus low AI rats, supporting a genetic role in the differences in gene expression that we observed. The correlation metrics obtained from our analysis were modest, as expected due to three limitations of the predictive model. First, the models are trained on whole-brain tissue lacking the cell-type-specific resolution of our snRNA-seq data. Third, the models can capture only a small fraction of variation in expression and do not account for trans-acting eQTLs or numerous other influences on gene expression. Despite these limitations, this analysis establishes that at least some of the differences are due to genetic variation Supplementary Fig. As more rat behavioral GWAS are completed, it will be possible to uncouple the role of genetics versus cocaine exposure more fully, for example, through the use of polygenic risk scores for addiction-related traits 28 , 30 , 31 , 32 , Consistent with previous findings showing enhanced GABAergic transmission following excessive cocaine use 81 , our differential gene expression analysis showed enrichment of genes in the GABAergic synapse pathway Fig. Moreover, we found that inhibition of GLO1—the enzyme responsible for MG metabolism—restored electrophysiological Fig. GABA A receptor agonists used in the context of cocaine-seeking behavior have shown contrasting results leading to both reductions and increases in cocaine-seeking behaviors 84 , 85 , 86 , 87 , 88 , 89 , Since MG is generated in proportion to glycolytic activity of nearly every cell and does not accumulate in synaptic vesicles, it may activate GABA A receptors at synaptic and extra synaptic sites; thus, manipulating the endogenous levels of MG by GLO1 inhibition represents a unique mechanism of GABA A receptor regulation. In our electrophysiological experiments, we did not observe changes in postsynaptic currents in the CeA; thus, we speculate that MG-based pharmacological manipulations may alter presynaptic GABA A receptor function, reducing GABA release at inhibitory terminals and suppressing inhibitory connections in the CeA. Consistent with this notion, previous studies have demonstrated that the activation of presynaptic GABA B receptors suppresses inhibitory connection in the CeA 91 and that negative regulation of GABAergic transmission can occur through a presynaptic mechanism An alternative scenario is that the magnitude of effects is not sufficient to cause detectable changes in amplitude. Overall, these results offer a new pharmacological target for improving therapeutic approaches for cocaine addiction. Previous studies manipulating GLO1 activity directly in the mouse amygdala by transgenic expression of Glo1 or MG microinjection were sufficient to reduce anxiety-like behaviors Future experiments targeting specific subregions or cell types of the amygdala will be necessary to further characterize the effects of GLO1 inhibition on cocaine addiction-related phenotypes. The results from the GLO1 inhibition experiments indicate that an altered metabolic state in the amygdala impacts several cellular processes that are involved in vulnerability to, and development of, addiction. Moreover, genes differentially regulated in high versus low AI rats were enriched in pathways related to energy metabolism, such as oxidative phosphorylation, which determines cellular ATP levels ATP is not only crucial for sustaining electrophysiological activity and cell signaling in the brain 95 , 96 , it is also required for ATP-dependent chromatin remodeling events initiated by pioneer TFs This could potentially explain the striking observations that excitatory and inhibitory neurons show opposite directions of regulation in chromatin accessibility Supplementary Fig. Future experiments that directly manipulate the expression of specific metabolic enzymes or pioneer TFs in a cell-type-specific manner will be necessary to fully elucidate their role in addiction. In conclusion, the amygdalar cellular atlas produced by this study is a valuable resource for understanding the role of cell-type-specific gene regulatory programs in the development of cocaine addiction-related behaviors. Our results emphasize the importance of cellular energetics and GABA A -mediated signaling in the enduring effects of cocaine use, and identify GLO1 as a potential new target for the treatment of cocaine addiction. To minimize inbreeding and control genetic drift, the HS rat colony is maintained using an optimized breeding strategy, with each breeder pair contributing one male and one female to subsequent generations. We used 46 HS rats for the behavioral experiments presented in Fig. Additionally, 26 of these 46 behaviorally phenotyped rats 13 female, 13 male were used for the cue-induced reinstatement experiments. We used a different cohort of 15 female and male HS rats 5 high AI, 5 low AI, 5 naive for the electrophysiology experiments. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications 99 , Behavioral data was collected with the MedPCIv v. Punches from three sections were combined for each rat. Briefly, frozen tissue was homogenized via glass dounce. Then, 12, nuclei were loaded onto a Chromium Controller 10x Genomics. For snATAC-seq libraries from the whole amygdala tissues, nuclei were purified using an established method Nuclei were isolated by iodixanol Millipore Sigma, catalog no. D density gradient. PN DNA was amplified for eight cycles. Nuclei were isolated as described above for snATAC-seq libraries. RNAse inhibitors Roche Diagnostics, catalog no. After the transposition reaction, nuclei were encapsulated and barcoded. Next-generation sequencing libraries were constructed following the User Guide. Final libraries were sequenced using the NovaSeq Illumina. Briefly, after surgical implantation of intravenous catheters and a week of recovery, HS rats were trained to self-administer cocaine fixed ratio 1 with 0. The breakpoint Fig. Rats were classified as high AI or low AI via a median split , AI was computed by averaging normalized measurements z -scores for the three behavioral tests after the LgA phase: escalation of drug intake, motivation and compulsive-like behavior, or drug taking despite adverse consequences Fig. For the pBBG studies Fig. Four weeks after the last IVSA session, the rats were placed back in the SA chambers without the availability of cocaine. The min timepoint was selected based on a previous study Data were analyzed using Prism v. For pairwise comparisons, data were analyzed using the unpaired t -test. Data distributions were assumed to be normal, but this was not tested formally. Experimenters were blinded to group allocation during behavioral data collection before brain collection. The frequency, amplitude and kinetics of sIPSCs were analyzed using semiautomated threshold-based minidetection software Easy Electrophysiology and confirmed visually. All snRNA-seq preprocessing was performed with Seurat v. We removed cells for which any of these metrics was more than three s. Next, we normalized the count data for each sample using sctransform with percent. We clustered the integrated dataset by constructing a K-nearest neighbor KNN graph using the first 30 principal components followed by the Louvain algorithm. To compare CeA and BLA subregion samples with the whole amygdala, we subsampled whole amygdala samples from the naive rats and performed the same integration technique. The integrated subregion data was visualized using UMAP. Cell type identities were assigned based on expression of known marker genes. We did not pre-filter genes for testing based on logFC or minimum fraction of cells in which a gene was detected. Permutation tests were performed using the same methods, covariates and filtering options but with shuffled AI labels. To obtain bootstrap distributions of DEG effect sizes, we resampled nuclei with replacement 1, times. Resampling was performed separately for nuclei from high and low AI rats so that the sample size of each set remained consistent. We then rescaled the coefficient to be in units of log2FC. Predicted relative expression was obtained for 26 rats with genotypes, for genes with at least one significant cis-eQTL. Genes with zero-variance predictions were removed, resulting in predictions for 8, genes. To estimate prediction accuracy, Pearson correlation r 2 was calculated between predicted and observed log-TPM expression for the rats used to discover whole-brain-hemisphere eQTLs. We removed cells where any of these metrics was more than two s. Each sample was normalized using term frequency-inverse document frequency followed by singular value decomposition The combined steps of term frequency-inverse document frequency followed by singular value decomposition are known as latent semantic indexing , We merged the data across all samples, repeated the process of latent semantic indexing and integrated the merged dataset using Harmony We observed successful reduction of batch effects following integration Supplementary Fig. Raw counts were used for downstream differential accessibility analyses. This uses the number of fragments per cell overlapping the promoter region of a given gene to calculate a gene activity score. Cell type identities were assigned by integrating the snATAC-seq data with the integrated snRNA-seq data and performing label transfer This process returns a classification score for each cell for each cell type defined in the scRNA-seq data. Each cell was assigned the cell-type identity with the highest score. Permutation tests were performed in the same manner as the differential gene expression analyses. Finally, using the baseline model and standard regression weights from the LDSC Partitioned Heritability tutorial, we ran a cell-type-specific partitioned heritability analysis. For the first FET, we used the annotatePeaks. We measured cell-type-specific motif activities using chromVAR to test for per motif deviations in accessibility between nuclei from high versus low AI rats. Differential analysis of chromVAR deviation scores was performed using the Wilcoxon rank-sum test between high versus low AI rats in each cell type. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. The following publicly available datasets were used: Rattus norvegicus Ensembl v. Janak, P. From circuits to behaviour in the amygdala. Nature , — Roberto, M. Cold Spring Harb. Med 2 , a Buffalari, D. Self, D. Koob, G. Pizzagalli, D. Pickens, C. Neurobiology of the incubation of drug craving. Trends Neurosci. Kalivas, P. The neural basis of addiction: a pathology of motivation and choice. Psychiatry , — Article PubMed Google Scholar. Kilts, C. Neural activity related to drug craving in cocaine addiction. Psychiatry 58 , — Aerts, T. Novel perspectives on the development of the amygdala in rodents. Ehrlich, I. Amygdala inhibitory circuits and the control of fear memory. Neuron 62 , — Ciocchi, S. Encoding of conditioned fear in central amygdala inhibitory circuits. Yao, Z. A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex. Di Bella, D. Molecular logic of cellular diversification in the mouse cerebral cortex. Ziffra, R. Single-cell epigenomics reveals mechanisms of human cortical development. Zhang, Z. Epigenomic diversity of cortical projection neurons in the mouse brain. Li, Y. An atlas of gene regulatory elements in adult mouse cerebrum. Domcke, S. A human cell atlas of fetal chromatin accessibility. Science , eaba Zhang, K. A single-cell atlas of chromatin accessibility in the human genome. Cell , — Tran, M. Single-nucleus transcriptome analysis reveals cell-type-specific molecular signatures across reward circuitry in the human brain. Neuron , — Chen, R. Decoding molecular and cellular heterogeneity of mouse nucleus accumbens. He, J. Transcriptional and anatomical diversity of medium spiny neurons in the primate striatum. Phillips, R. An atlas of transcriptionally defined cell populations in the rat ventral tegmental area. Cell Rep. Avey, D. Savell, K. A dopamine-induced gene expression signature regulates neuronal function and cocaine response. Carrette, L. The cocaine and oxycodone biobanks, two repositories from genetically diverse and behaviorally characterized rats for the study of addiction. Chen, B. Rescuing cocaine-induced prefrontal cortex hypoactivity prevents compulsive cocaine seeking. Cohen, A. Robust escalation of nicotine intake with extended access to nicotine self-administration and intermittent periods of abstinence. Neuropsychopharmacology 37 , — Addiction as a stress surfeit disorder. Neuropharmacology 76 , — Solberg Woods, L. Using heterogeneous stocks for fine-mapping genetically complex traits. Methods Mol. Hansen, C. Development of the National Institutes of Health genetically heterogeneous rat stock. Alcohol Clin. Saar, K. SNP and haplotype mapping for genetic analysis in the rat. Baud, A. Combined sequence-based and genetic mapping analysis of complex traits in outbred rats. Leptin protects against the development and expression of cocaine addiction-like behavior in heterogeneous stock rats. Large-scale characterization of cocaine addiction-like behaviors reveals that escalation of intake, aversion-resistant responding, and breaking-points are highly correlated measures of the same construct. Sedighim, S. Individual differences in addiction-like behaviors and choice between cocaine versus food in heterogeneous stock rats. George, O. Extended access to cocaine self-administration produces long-lasting prefrontal cortex-dependent working memory impairments. Neuropsychopharmacology 33 , — Butler, A. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Zeisel, A. Molecular architecture of the mouse nervous system. Saunders, A. Molecular diversity and specializations among the cells of the adult mouse brain. Tasic, B. Shared and distinct transcriptomic cell types across neocortical areas. Nature , 72—78 A multimodal cell census and atlas of the mammalian primary motor cortex. Nature , 86— Extensive and spatially variable within-cell-type heterogeneity across the basolateral amygdala. Beyeler, A. Neuronal diversity of the amygdala and the bed nucleus of the stria terminalis. Leek, J. Tackling the widespread and critical impact of batch effects in high-throughput data. Tran, H. A benchmark of batch-effect correction methods for single-cell RNA sequencing data. Genome Biol. Munro, D. The regulatory landscape of multiple brain regions in outbred heterogeneous stock rats. Nucleic Acids Res. Tsuboi, D. Dopamine drives neuronal excitability via KCNQ channel phosphorylation for reward behavior. Hansen, H. The neuronal KCNQ channel opener retigabine inhibits locomotor activity and reduces forebrain excitatory responses to the psychostimulants cocaine, methylphenidate and phencyclidine. Cruz, B. FKBP5 inhibitors modulate alcohol drinking and trauma-related behaviors in a model of comorbid post-traumatic stress and alcohol use disorder. Neuropsychopharmacology 48 , — Levran, O. Stress-related genes and heroin addiction: a role for a functional FKBP5 haplotype. Psychoneuroendocrinology 45 , 67—76 Heller, E. Morphine and cocaine increase serum- and glucocorticoid-inducible kinase 1 activity in the ventral tegmental area. Mohammadi, P. Quantifying the regulatory effect size of cis-acting genetic variation using allelic fold change. Genome Res. Subramanian, A. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Natl Acad. USA , — Kasischke, K. Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science , 99— Attwell, D. An energy budget for signaling in the grey matter of the brain. Blood Flow. Distler, M. Perez, C. A metal-binding pharmacophore library yields the discovery of a glyoxalase 1 inhibitor. Inhibition of glyoxalase 1 reduces alcohol self-administration in dependent and nondependent rats. Zhang, Y. Robinson, M. Bioinformatics 26 , — Love, M. Schep, A. Methods 14 , — Matsuda, T. Pioneer factor neuroD1 rearranges transcriptional and epigenetic profiles to execute microglia-neuron conversion. Glahs, A. Putting chromatin in its place: the pioneer factor NeuroD1 modulates chromatin state to drive cell fate decisions. EMBO J. Cruz, F. Using c-fos to study neuronal ensembles in corticostriatal circuitry of addiction. Brain Res. Overexpression of DeltaFosB in nucleus accumbens mimics the protective addiction phenotype, but not the protective depression phenotype of environmental enrichment. Bali, P. Transcriptional mechanisms of drug addiction. Dialogues Clin. Walker, D. Psychiatry 84 , — Nestler, E. USA 98 , — Hope, B. Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments. Neuron 13 , — Nye, H. Induction of chronic Fos-related antigens in rat brain by chronic morphine administration. Pharmacological studies of the regulation of chronic FOS-related antigen induction by cocaine in the striatum and nucleus accumbens. Moratalla, R. Network-level changes in expression of inducible Fos—Jun proteins in the striatum during chronic cocaine treatment and withdrawal. Neuron 17 , — Bulik-Sullivan, B. LD score regression distinguishes confounding from polygenicity in genome-wide association studies. Liu, M. Association studies of up to 1. Polimanti, R. Leveraging genome-wide data to investigate differences between opioid use vs. Psychiatry 25 , — Wade, C. Compulsive-like responding for opioid analgesics in rats with extended access. Neuropsychopharmacology 40 , — Belin, D. Responses to novelty and vulnerability to cocaine addiction: contribution of a multi-symptomatic animal model. Visscher, P. Discovery and implications of polygenicity of common diseases. Science , — Zaret, K. Pioneer transcription factors initiating gene network changes. Genet 54 , — Chitre, A. Genome wide association study in 3, outbred rats identifies multiple loci for body weight, adiposity, and fasting glucose. Obesity Silver Spring 28 , — Kallupi, M. Kappa opioid receptor-mediated dysregulation of GABAergic transmission in the central amygdala in cocaine addiction. Psychiatry 74 , — Stephens, D. GABAA receptor subtype involvement in addictive behaviour. Genes Brain Behav. McMurray, K. Identification of a novel, fast acting GABAergic anti-depressant. Psychiatry 23 , — Bentzley, B. Inhibiting subthalamic nucleus decreases cocaine demand and relapse: therapeutic potential. Shinohara, F. The role of dopaminergic signaling in the medial prefrontal cortex for the expression of cocaine-induced conditioned place preference in rats. Mitchell, S. Neuropharmacology , 98— Sun, W. Psychopharmacology Berl. Pelloux, Y. Opposite effects of basolateral amygdala inactivation on context-induced relapse to cocaine seeking after extinction versus punishment. Madangopal, R. Inactivation of the infralimbic cortex decreases discriminative stimulus-controlled relapse to cocaine seeking in rats. Neuropsychopharmacology 46 , — Cruz, A. Prelimbic cortical projections to rostromedial tegmental nucleus play a suppressive role in cue-induced reinstatement of cocaine seeking. Delaney, A. Baclofen acts in the central amygdala to reduce synaptic transmission and impair context fear conditioning. Li, C. Psychiatry 71 , — Neuronal overexpression of Glo1 or amygdalar microinjection of methylglyoxal is sufficient to regulate anxiety-like behavior in mice. Brain Res , — Boyer, P. What makes ATP synthase spin? Du, F. Tightly coupled brain activity and cerebral ATP metabolic rate. ATP and brain function. Blood Flow Metab. Swinstead, E. Pioneer factors and ATP-dependent chromatin remodeling factors interact dynamically: a new perspective. Bioessays 38 , — Finucane, H. Partitioning heritability by functional annotation using genome-wide association summary statistics. Nociceptin attenuates the escalation of oxycodone self-administration by normalizing CeA—GABA transmission in highly addicted rats. Da Mesquita, S. Lepack, A. Dopaminylation of histone H3 in ventral tegmental area regulates cocaine seeking. Fulton, S. Histone H3 dopaminylation in ventral tegmental area underlies heroin-induced transcriptional and behavioral plasticity in male rats. Neuropsychopharmacology 47 , — Werner, C. Ubiquitin-proteasomal regulation of chromatin remodeler INO80 in the nucleus accumbens mediates persistent cocaine craving. Neuroadaptations in the dorsal hippocampus underlie cocaine seeking during prolonged abstinence. Calipari, E. Synaptic microtubule-associated protein EB3 and SRC phosphorylation mediate structural and behavioral adaptations during withdrawal from cocaine self-administration. Carpenter, M. Nr4a1 suppresses cocaine-induced behavior via epigenetic regulation of homeostatic target genes. Duttke, S. Glucocorticoid receptor-regulated enhancers play a central role in the gene regulatory networks underlying drug addiction. Deroche-Gamonet, V. Evidence for addiction-like behavior in the rat. Pattern of intake and drug craving predict the development of cocaine addiction-like behavior in rats. Psychiatry 65 , — Satpathy, A. Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion. Zheng, G. Massively parallel digital transcriptional profiling of single cells. Cunningham, F. Ensembl Castro-Mondragon, J. Stuart, T. Comprehensive integration of single-cell data. Hafemeister, C. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Richards, L. A comparison of data integration methods for single-cell RNA sequencing of cancer samples. Finak, G. MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Wu, T. Innovation 2 , Single-cell chromatin state analysis with Signac. Methods 18 , — Quinlan, A. BEDTools: a flexible suite of utilities for comparing genomic features. Team TBD. R package version 1. Cusanovich, D. Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Deerwester, S. Indexing by latent semantic analysis. Article Google Scholar. Korsunsky, I. Fast, sensitive and accurate integration of single-cell data with Harmony. Methods 16 , — Yirga, A. Negative binomial mixed models for analyzing longitudinal CD4 count data. Purcell, S. PLINK: a tool set for whole-genome association and population-based linkage analyses. Heinz, S. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Cell 38 , — Download references. We thank S. We thank J. Hightower for assistance with figure preparation. We thank P. Montilla-Perez, L. Maturin and P. Schweitzer for technical assistance with sample collection and equipment maintenance. We thank L. Solberg Woods for HS rats breeding colony management. U01DA to F. Jessica L. Zhou, Aaron J. Chitre, Daniel Munro, Lieselot L. Carrette, Olivier George, Abraham A. You can also search for this author in PubMed Google Scholar. The inventors of this patent are A. Palmer and M. All other authors declare no competing interests. Per nucleus metrics for all nuclei in snRNA-seq dataset after filtering. This table contains selected columns from the metadata table for the Seurat object containing the integrated snRNA-seq data. Per nucleus metrics for all nuclei in snATAC-seq dataset after filtering. This table contains selected columns from the metadata table for the Signac object containing the integrated snATAC-seq data. All cell-type-specific differential gene expression analysis results, obtained using the negative binomial test. Results of permutation test for differential gene expression analysis using negative binomial test. All cell-type-specific differential peak accessibility analysis results, obtained using the negative binomial test. Permutation test for differential peak accessibility analysis results using negative binomial test. Reprints and permissions. Zhou, J. Single-nucleus genomics in outbred rats with divergent cocaine addiction-like behaviors reveals changes in amygdala GABAergic inhibition. Nat Neurosci 26 , — Download citation. Received : 03 September Accepted : 06 September Published : 05 October Issue Date : November Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Skip to main content Thank you for visiting nature. Download PDF. This article has been updated. Abstract The amygdala processes positive and negative valence and contributes to addiction, but the cell-type-specific gene regulatory programs involved are unknown. Methamphetamine-induced region-specific transcriptomic and epigenetic changes in the brain of male rats Article Open access 27 September Cell-type specific transcriptional adaptations of nucleus accumbens interneurons to amphetamine Article 16 February Key transcription factors mediating cocaine-induced plasticity in the nucleus accumbens Article 02 June Main The amygdala regulates numerous behaviors related to emotions, motivation and memory 1 and is implicated in various neuropsychiatric disorders including addiction 2 , 3. Full size image. Discussion To better understand the molecular basis of addiction, we generated an atlas of single-cell gene expression and chromatin accessibility in the amygdala of rats with divergent cocaine addiction-like behaviors after a prolonged period of abstinence. Integrating snATAC-seq data across samples and clustering Each sample was normalized using term frequency-inverse document frequency followed by singular value decomposition Partitioned heritability analysis We downloaded summary statistics for the GWAS of tobacco and alcohol use by Liu et al. Measuring differential activity of TFs with chromVAR We measured cell-type-specific motif activities using chromVAR to test for per motif deviations in accessibility between nuclei from high versus low AI rats. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. References Janak, P. Article Google Scholar Korsunsky, I. Acknowledgements We thank S. Palmer Authors Jessica L. Zhou View author publications. View author publications. Ethics declarations Competing interests A. Supplementary information. Supplementary Information Supplementary Figs. Reporting Summary. Supplementary Data 5 All cell-type-specific differential gene expression analysis results, obtained using the negative binomial test. Supplementary Data 6 Results of permutation test for differential gene expression analysis using negative binomial test. Supplementary Data 9 All cell-type-specific differential peak accessibility analysis results, obtained using the negative binomial test. Supplementary Data 10 Permutation test for differential peak accessibility analysis results using negative binomial test. Supplementary Data 12 ChromVar analysis results. About this article. Cite this article Zhou, J. Copy to clipboard. This article is cited by Role of serotonin neurons in the dorsal raphe nucleus in heroin self-administration and punishment Chen Li Nicholas S. McCloskey Lynn G. Kirby Neuropsychopharmacology Search Search articles by subject, keyword or author. Show results from All journals This journal. Advanced search. Close banner Close. Email address Sign up. Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing.

A Garbage Bag in Paradise

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Mike and I were crammed in the way-back, the provisional row of seats in the trunk, and the rhythmic, metallic clang was striking directly below our feet. We looked at each other, concluding the dismal inevitable. Midway up a tortuous hill, the jeep gave a terrible, final clank and stopped, and our driver shook his head sadly. We stood on the roadside in the jungle. Soon the boorish hooting of howler monkeys began nearby, an intimidating sound, which is its purpose, since howlers defend their turf with jeering boos. He seemed confident the situation would be speedily resolved, for every few minutes a car or jeep would hurtle around the visible bends of the solitary road. The young French couple with a plane to catch were awarded the first two open seats. Of all things, I found myself thinking about the trash bag I had stashed in the jeep. Now I was wondering—should I transfer the trash bag into the next jeep, or leave it in our busted ride? But what was I going to do with it back in Panama City? On the other hand, if I left it in the abandoned jeep, the noble gesture became pointless, even more futile than it already was. A token act of environmentalism, but I would see it though. This was at the end of our two-week trip through Panama. Back in Boulder we were roommates, renting a rustic ranch house on a horse farm a few miles from town. Ours is an intimate, old-fashioned friendship. When we travel together, we try to follow our instinct, seeking a kind of pure experience, the reality of a place, rather than any proscribed route. Panama is an isthmus—a wave-shaped strip of land between the Pacific to the south and the Carribean to the north—connecting Costa Rica to Colombia like the root of an umbilical cord. She was tanned and straw-haired with an appealing Joni Mitchell thing going and a Midwestern rambling manner. He had to bribe the police to avoid jail and now he was under their thumb, on precarious parole. Mike and I were fine with cheap local beer and a bit of the grungy pot Karen liked to smoke from a perforated apple. That evening, sitting on the veranda of her apartment which hung on a cliff above a secluded crescent of rugged Pacific coastline, I could feel euphoria sifting into my system. We had escaped the hectic capital; it felt like we had finally arrived in Panama, the place I had imagined, laidback and idyllic. Motionless geckos on the ceiling chirped to each other, surprisingly loud tongue-clucking sounds from such tiny conversationalists. The waves below crumpled and hissed in retreat. In the morning we watched pelicans dive-bomb for fish in the bay. The giant birds hovered in the humid sky, cruising the updrafts, scanning the surface, then gave into gravity and plunged like a squadron of kamikaze into the waves, bobbing upright, swallowing their catch. Mike and I swam out to see them up close, their long sword-like bills and yellow-feathered heads. The water had a delicious chill and reeked of chum. A good life, it seemed, retirement in this sleepy pacific paradise. Cheap rent, fresh fish and produce, and if you apply for and acquire a visa, heavy discounts on accommodation and transport around the country. A growing number of gringos appear to agree. They all wore the contentment of being in on a luxurious secret. Private gated communities with names like Bella Mar and half-constructed mansions had already sprung up along the pitted coastal road. The massive photo displayed a slender beauty from the bikinied buttocks down, a surfboard leash around her ankle, strolling toward the limitless blue shimmer. Regardless, the way was irrefutable, and depressingly familiar. A similar transformation was occurring in Casco Viejo, the up-and-coming neighborhood in the capital. Spanish colonial flair still lingered on the narrow streets in the rippled red rooftiles and wrought-iron verandas, but half the housefronts were crumbled into decrepitude. Heavy renovation was happening, scaffolds climbing dilapidated lanes, rows of architectural ruins converting into sleek avenues of gelato shops and boutique hotels and tapas restaurants, drawn in creamy pastels like parts of Havana. At night dolled-up partygoers trawled the main square in their cars while outdoor cafes blasted reggaeton; from the veranda of our hostel we could see a rooftop hotel bar encased in glass a la Miami. Casco Viejo was becoming uber-hip, perhaps too quickly for its own good. The district was situated on a small fortified peninsula, and the prow-like harbor wall looked over the hazy bay to the astonishing skyline of downtown Panama City, bristling with spires, company monoliths, residential towers, skeletal works-in-progress, a high-concept skyscraper of spiraling green glass that belongs in Dubai—a vision of the future from the ramparts of the old world. Along the Pan-American highway we rode through clouds of red construction dust, past caterpillars and backhoe loaders chewing red earth from a cross-section of hillside, small armies of workers and traffic coordinators wearing bandanas over their mouths. Panama is taking command of its destiny, after a bloody history of bombardment, oppression, and interference by Spain, France, and the United States, not to mention their own malignant rulers, like Noriega. Control of the lucrative Canal was finally ceded to Panama in and fully assumed by the government in , and plans are underway for its expansion. More traffic, more infrastructure, more money, more tourists, more displacement of locals, more upward mobility for the few. Who am I to judge this inevitable trend, to find it discomforting—me, the privileged, visiting American? In Venao after dark, Mike and I stumbled upon a wedding party on the beach. The celebrants were dressed in white and setting Chinese sky lanterns adrift into the night: oil-paper orbs with fires lit inside which carried them upward like hot-air balloons, up and up until they were luminous blobs against the blackness. It was beautiful to watch, the white-garbed clique holding the lanterns aloft like worshippers, the levitating balls of orange flame lifting from their fingers. But what happens when they come down, I wondered. Afloat in the ocean it becomes a perfect net to entangle a tortoise or dolphin. Or a perfect firestarter, if the wind hooks it inland. A perfect metaphor, too, for the unintended consequences of it all: releasing fireballs into the dark for the oohs and ahs, the picturesque ephemerality, the unseen descent. My good-humored misanthropy became more focused, more aware of human impact on the planet, the rise in population and spread of homogenized culture. Mike and I discussed it often back home: the blind denial of climate change, damaging power of greed, presumption of ownership over the earth. We would sigh, wearily resigned to the doom of our species, and then toast to being alive as the end of the party draws nigh. But I see how deliberate obliviousness happens, abroad and at home. Yet what still exists holds more species biodiversity than any other New World country north of Colombia, and from lookout points in the highlands, the jungle seems boundless. Curtains of deliquescent mist down from the mossy mountains where icebergs of cloud pile and drift in their valleys. How could anyone worry about forest devastation in the midst of such splendor? Industrialization, deforestation, trash accumulation—there was nothing I could do about these things. The Guna are the first Latin American indigenous people to gain autonomy of their comarca , after a violent revolt in Crossing into their territory was like permeating a lax international boundary. A Spaniard in the jeep next to me murmured about the booming cocaine trade that runs through the region from Colombia, just down the coast, a popular route for sailing tours and drug-runners. The isles closer to shore were more populated and packed with ramshackle bamboo huts and corrugated metal structures leaning crookedly over the lapping waterline. They looked like an overburdened flotilla slowly sinking. We had arranged to stay on Chichime, one of the larger outlying isles, covered in coconut groves with a few thatch-roofed huts. Mike and I were given a hut all to ourselves: two beds, a table, a sand floor, slats of light piercing the wicker-lashed walls, the patchwork sapphire-and-emerald water twenty feet away. He was Guna, an elder of the tiny group that ran the island. Like the other Guna men he seemed barely above five feet tall, leathered and sinewy. At last he said, with a studied offhandedness, like an afterthought, that if we were going to smoke marijuana, we should go off in the trees. I live just over there, he told us, pointing vaguely; he must have caught the smoke on the breeze. We nodded, vaguely chastened—absolutely, we agreed. He looked at us with his canny, aged face. After a surprised pause, we shook our heads. No, not us, no thank you. We demurred, and he shrugged and nodded, and wandered away. Half a dozen other travelers were staying on the island. The Guna attitude toward us all was curious. The women cooked and served meals; they wore bright woven molas , patterned fabrics, and deadpan expressions. The food was plonked down on the table with little love or fanfare: simple dishes of rice, salad, and fish. The fish was fresh but often cold, as if left sitting out, and unseasoned, and the women did not respond to our hearty gracias. Their stone-faced manner was perhaps a defensive response to the picture-snapping hordes off yachts and cruise ships that regularly stopped here. All these visitors, daytrippers and overnighters, generate garbage, of course. And the shoreline of the island all the way around was ringed with washed-up detritus: plastic bottles, beer cans, styrofoam containers, discarded flipflops, shredded clothing, fronds of dead coral. Bags of trash left behind by daytrippers sat in the palm grove for days, blown about by the wind. We heard rumors of the other way the Guna disposed of trash. The sailor had been warned to refuse, since the Guna supposedly ferry the trash out to sea and dump it. The tide pulls away and redeposits the flotsam on neighboring islands until all are equally adorned. Mike and I picked our way around the littered shore, looking at crabs, starfish, Fresca bottles, Soberana beercans, something that resembled a diaper, while a fleet of sailboats cluttered the view of the neighboring island and a cruise liner hulked like an office building farther asea. In my imagination I erased it all, and tried to envision this place a hundred years ago. A timeless scene, the Guna paddling dugout canoes, the shallow harbor bristling with healthy coral, the shoreline strewn only with coconuts and palm fronds. From their perspective, they must have other, more important fish to fry, like organizing and hosting the legions of backpackers who show up every day, more and more every year, armed with their cosmopolitan expectations of paradise. So there I was, transferring the plastic bag into the jeep which had rescued us from the side of the road in the jungle. I was starting to feel carsick, myself. There was no armrest, nothing to hold onto as the Land Cruiser careened around the steep bends. And a warm, sordid odor was beginning to waft from behind me: a saturnalian frathouse stink of beer dregs and rotting banana peels and chorizo wrappers. Our trash bag, undeniable, inescapable, was riding atop the luggage directly behind my head. This was perfect justice, a well-deserved reminder of the futility of our petty gesture. For what were we going to do with the garbage back in Panama City? Leave it on the curb, alongside the bins of our hotel, to be taken god-knows-where? Human beings love beauty, above almost all else. But our love is death to natural beauty, for where we go, pollution follows. The act of traveling itself, hopping on an airplane, is an insidious contributor to climate change; a seven-hour flight dumps two to three tons of carbon dioxide emissions into the atmosphere per passenger. And as Panama expands its canal, pretties up its cities, lures expats into ritzy beachfront developments, more tourists will arrive every year, a simple mathematical formula. On the one hand, of course we should go, for travel is also what defines our species, and we must live our lives. Why walk when we can fly, has been our credo. Yet we face forward when we fly, never truly looking ahead, and the wake of our descent is always unseen. Created by Grove Atlantic and Electric Literature. Sign Up For Our Newsletters. How to Pitch Lit Hub. Advertisers: Contact Us. Privacy Policy. For the past decade, Literary Hub has brought you the best of the book world for free—no paywall. But our future relies on you. By Daniel Levine. Article continues after advertisement. Remove Ads. 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